escherichia coli exonuclease New England Biolabs Search Results


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  • 95
    New England Biolabs exonuclease i
    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following <t>Exonuclease</t> I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    Exonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 3810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs dna polymerase i
    Primer extension activity of exonuclease-deficient Klenow fragment of <t>DNA</t> <t>polymerase</t> I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.
    Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs exonuclease iii
    Probing of the EC translocation conformations. ( A ) <t>Exo</t> <t>III</t> footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).
    Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exonuclease iii e coli
    Probing of the EC translocation conformations. ( A ) <t>Exo</t> <t>III</t> footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).
    Exonuclease Iii E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs bal31 nuclease
    Probing of the EC translocation conformations. ( A ) <t>Exo</t> <t>III</t> footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).
    Bal31 Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t5 exonuclease
    Probing of the EC translocation conformations. ( A ) <t>Exo</t> <t>III</t> footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).
    T5 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 523 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs λ exonuclease
    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with <t>λ</t> exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).
    λ Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs 10x exonuclease i reaction buffer
    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with <t>λ</t> exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).
    10x Exonuclease I Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t7 exonuclease
    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with <t>T7</t> exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.
    T7 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    New England Biolabs exonuclease 1 exo1
    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with <t>T7</t> exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.
    Exonuclease 1 Exo1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 87/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    New England Biolabs exonuclease 1 recombinant shrimp alkaline phosphatase
    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with <t>T7</t> exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.
    Exonuclease 1 Recombinant Shrimp Alkaline Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 83/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Journal: PLoS ONE

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    doi: 10.1371/journal.pone.0016925

    Figure Lengend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Article Snippet: In cases where the COLIGO was still contaminated by > 5% of the linear oligonucleotide after elution (as determined by gel staining), an Exonuclease I (NEB) digest was done.

    Techniques: Polyacrylamide Gel Electrophoresis

    Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.

    Journal: Nucleic Acids Research

    Article Title: DNA-protein cross-linking by trans-[PtCl2(E-iminoether)2]. A concept for activation of the trans geometry in platinum antitumor complexes

    doi: 10.1093/nar/gkg863

    Figure Lengend Snippet: Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ). ( A ) Experiments were conducted using the 8mer/23mer primer/template duplex for various times using undamaged template (lanes 1–5), the template containing monofunctional adduct of [Pt(dien)Cl]Cl (lanes 6–10), monofunctional adduct of trans-EE (lanes 11–15) or 1,2-GG intrastrand CL of cisplatin (lanes 16–20). Timings were as follows: 1 min, lanes 1, 6, 11 and 16; 3 min, lanes 2, 7, 12 and 17; 15 min, lanes 3, 8, 13 and 18; 30 min, lanes 4, 9, 14 and 19; 60 min, lanes 5, 10, 15 and 20. The pause sites opposite the platinated guanines and flanking residues are marked 12, 13 and 14 (the sites opposite the platinated residues are still marked ‘Pt’). The nucleotide sequences of the templates and the primer are shown beneath the gels. ( B ) The time dependence of the inhibition of DNA synthesis on undamaged (control) template (open circles), DNA containing monofunctional adduct of [Pt(dien)Cl]Cl (closed triangles), DNA containing monofunctional adduct of trans-EE (open squares) or DNA containing 1,2-GG intrastrand CL of cisplatin (closed circles). Data are means (±SE) from three different experiments with two independent template preparations.

    Article Snippet: T4 DNA ligase, the Klenow fragment from DNA polymerase I (exonuclease minus, mutated to remove the 3′→5′ proofreading domain) (KF– ), restriction endonuclease EcoRI and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Inhibition, DNA Synthesis

    Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ) ( A ) and RT HIV-1 ( B ) using the 8mer/40mer and 17mer/30mer primer/template duplexes, respectively. The experiments were conducted for 30 min using undamaged templates (lanes 1), undamaged templates to which histone H1 was added at a molar ratio of 4:1 (lanes 2), the templates containing monofunctional adduct of trans-EE (lanes 3) and monofunctional adduct of trans-EE cross-linked to histone H1 (lanes 4). The pause sites opposite the platinated guanines and flanking residues are marked 19, 20, 21 and 22 (the sites opposite the platinated residue are still marked ‘Pt’). The nucleotide sequences of the templates and the primers are shown beneath the gels.

    Journal: Nucleic Acids Research

    Article Title: DNA-protein cross-linking by trans-[PtCl2(E-iminoether)2]. A concept for activation of the trans geometry in platinum antitumor complexes

    doi: 10.1093/nar/gkg863

    Figure Lengend Snippet: Primer extension activity of exonuclease-deficient Klenow fragment of DNA polymerase I (KF – ) ( A ) and RT HIV-1 ( B ) using the 8mer/40mer and 17mer/30mer primer/template duplexes, respectively. The experiments were conducted for 30 min using undamaged templates (lanes 1), undamaged templates to which histone H1 was added at a molar ratio of 4:1 (lanes 2), the templates containing monofunctional adduct of trans-EE (lanes 3) and monofunctional adduct of trans-EE cross-linked to histone H1 (lanes 4). The pause sites opposite the platinated guanines and flanking residues are marked 19, 20, 21 and 22 (the sites opposite the platinated residue are still marked ‘Pt’). The nucleotide sequences of the templates and the primers are shown beneath the gels.

    Article Snippet: T4 DNA ligase, the Klenow fragment from DNA polymerase I (exonuclease minus, mutated to remove the 3′→5′ proofreading domain) (KF– ), restriction endonuclease EcoRI and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay

    Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).

    Journal: Nucleic Acids Research

    Article Title: Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations

    doi: 10.1093/nar/gkl559

    Figure Lengend Snippet: Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).

    Article Snippet: Exonuclease III (Exo III) (NEB, 0.1 U/μl) was added for 5–10 min at 37°C.

    Techniques: Translocation Assay, Incubation, Labeling, Irradiation, Nucleic Acid Electrophoresis

    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).

    Journal: Analytical biochemistry

    Article Title: Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA

    doi: 10.1016/j.ab.2008.06.037

    Figure Lengend Snippet: (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).

    Article Snippet: A mixture of supercoiled (3.7 µg) and linear (3.3 µg) DNA was incubated with λ exonuclease (1 µL, 5 units/µL) and RecJf (3 µL, 30 units/µL) in a total volume of 100 µL containing 1X λ exonuclease buffer (New England Biolabs) at 37 °C for 16 h. The nicked-circular DNA preparation (14 µg) containing traces of linear DNA was then incubated again with λ exonuclease (0.5 µL) and RecJf (3 µL) in a total volume of 100 µL containing 1X λ exonuclease buffer at 37 °C for 16 h. Next, the λ exonuclease was heat-inactivated (65 °C, 10 min), and the reaction mixture was extracted with phenol and chloroform:amyl alcohol (24:1, v/v).

    Techniques: Agarose Gel Electrophoresis, Incubation, Marker

    Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.

    Journal: Analytical biochemistry

    Article Title: Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA

    doi: 10.1016/j.ab.2008.06.037

    Figure Lengend Snippet: Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.

    Article Snippet: A mixture of supercoiled (3.7 µg) and linear (3.3 µg) DNA was incubated with λ exonuclease (1 µL, 5 units/µL) and RecJf (3 µL, 30 units/µL) in a total volume of 100 µL containing 1X λ exonuclease buffer (New England Biolabs) at 37 °C for 16 h. The nicked-circular DNA preparation (14 µg) containing traces of linear DNA was then incubated again with λ exonuclease (0.5 µL) and RecJf (3 µL) in a total volume of 100 µL containing 1X λ exonuclease buffer at 37 °C for 16 h. Next, the λ exonuclease was heat-inactivated (65 °C, 10 min), and the reaction mixture was extracted with phenol and chloroform:amyl alcohol (24:1, v/v).

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Marker

    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    doi: 10.1074/jbc.M109.025825

    Figure Lengend Snippet: BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Article Snippet: G-strand overhang was also measured after incubation of genomic DNA with T7 exonuclease (1 unit/μg DNA) (New England Biolabs) in NE-Buffer 4 at 25 °C for 60 s. T7 exonuclease acts in the 5′–3′ direction to remove 5′ nucleotides from duplex DNA.

    Techniques: Hybridization, Labeling, Transfection, Plasmid Preparation, Western Blot, Over Expression